Methods of Making Implantable Collagen Devices

ABSTRACT

The invention relates to implantable collagen devices made by seeding at least one elongate collagen construct, e.g., comprising at least one elongate synthetic collagen fiber with a plurality of cells and applying a strain and/or stress to the at least one elongate collagen fiber to induce the cells to differentiate into target phenotypes, e.g., tendon or ligament phenotype cells (and/or fibroblasts), typically with an extracellular matrix of collagen to organize into a tissue on the at least one collagen fiber.

RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.13/667,507, filed Nov. 2, 2012, which claims the benefit of and priorityto U.S. Provisional Patent Application Ser. No. 61/554,645, filed Nov.2, 2011, the contents of which are hereby incorporated by reference asif recited in full herein.

FIELD OF THE INVENTION

The invention relates to implantable collagen fiber devices and methodsof making the same.

BACKGROUND OF THE INVENTION

Surgical repair or replacement of injured or diseased tissues, such astendons and ligaments, often relies on the use of autologous tissue.However, an injury resulting from the harvesting of the autologoustissue can result in cell morbidity at a donor site.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the invention are directed to collagen-based materials,typically comprising elongate synthetic collagen fibers, withdifferentiated cells and/or an extracellular matrix of collagen.

A first aspect of the present invention is directed to implantablecollagen fiber constructs. The constructs include a plurality ofelongate collagen fibers and a plurality of cells attached to theelongate collagen fibers. The attached cells include tendon or ligamentphenotype cells or the attached cells include tendon or ligamentphenotype cells and an extracellular matrix that includes collagen. Thecollagen in the extracellular matrix is present in an amount greaterthan other extracellular matrix proteins.

The plurality of elongate collagen fibers can be arranged as constructbody. The tendon or ligament phenotype cells and extracellular matrixthat includes (predominantly) collagen can be organized into tendon-likeor ligament-like tissue that increases a volume of the construct body,measured dry, by between about 20-200% or that increases a thickness ofthe construct body by between 10%-200%.

The plurality of elongate collagen fibers can be arranged as a constructbody. The tendon or ligament phenotype cells and extracellular matrix ofcollagen can extend over at least 50% of a length of the construct bodyshape about the elongate collagen fibers.

The fibers can include tendon phenotype cells and at least 20%extracellular matrix of collagen by volume, dry.

The plurality of elongate collagen fibers define a construct body with ashape and the attached cells includes the tendon or ligament phenotypecells and the extracellular matrix of collagen, in an amount betweenabout 20% to 200% by volume of the construct body, dry.

The collagen fibers can be cross-linked with NDGA and the attachedtendon or ligament phenotype cells extend over at least a major portionof a surface area of each collagen fiber for at least 50% of an overalllength of the collagen fibers.

The plurality of collagen fibers can be wound, twisted, braided and/orwoven together.

The implantable collagen fiber construct can be a tendon or a ligamentprosthesis and the collagen fiber construct can have a tensile strength,stiffness, and dynamic flexibility that meets or exceeds that of anatural tissue the collagen construct is designed to mimic.

The attached cells are differentiated cells derived from cells selectedfrom the group consisting of embryonic, neonatal or adult cells,pluripotent stem cells from any tissue source, mesenchymal stem cells,tendon and/or ligament fibroblasts, or combinations thereof.

The plurality of elongate collagen fibers can be arranged in a constructbody shape, and the cells can be organized into tendon-like orligament-like tissue that increases a thickness of the construct bodybetween 100%-200%.

Other aspects are directed to methods of processing an implantablecollagen fiber(s). The methods include: (a) placing at least oneelongate collagen fiber in a desired orientation in a tissue culturevessel; (b) seeding the at least one elongate collagen fiber with aplurality of cells while in the vessel; and (c) automatically applying astrain and/or stress to the at least one elongate collagen fiber in thevessel to cause at least one of the following cellular actions: (i)induce the cells to differentiate into tendon or ligament fibroblasts ortendon or ligament phenotype cells; (ii) form an extracellular matrix ofsome or at least a major portion of collagen; or (iii) both induce thecells to differentiate into tendon or ligament fibroblasts or tendon orligament phenotype cells and form an extracellular matrix, wherein theextracellular matrix comprises collagen in amount greater than otherproteins.

The method can also include (d) producing an implantable collagen fiberconstruct with (i) the tendon or ligament phenotype differentiated cellsor (ii) the tendon or ligament phenotype differentiated cells and thecollagen based extracellular matrix based, at least in part, on theapplying step.

The tissue culture vessel can include a flexible pouch that holds the atleast one collagen fiber in the desired orientation during the applyingstep.

The seeding step can be carried out by introducing a fluid comprisingthe plurality of cells into the pouch via a fluid port of the flexiblepouch.

The cells of the seeding step can be selected from the group consistingof embryonic, neonatal or adult cells, pluripotent stem cells from anytissue source, mesenchymal stem cells, tendon and/or ligamentfibroblasts, or combinations thereof.

The seeding step can be carried by immersing or submerging the at leastone elongate collagen fiber into a fluid contained in the flexiblepouch. The fluid comprises the plurality of cells (typically as a cellsolution or suspension).

The applying step can be carried out by attaching a translatingmechanical tensioner to opposing end portions of the flexible pouch.

The applying step can be carried out at least periodically afterseeding.

The applying step can be carried out after a time sufficient to allowseed attachment to the at least one fiber, then at least periodicallyuntil implantation.

The applying step can be carried out to first apply a static strain orstress, then apply a cyclic strain or stress.

The applying step can be carried out to apply a static initial offsetstrain to the at least one elongate collagen fiber after the seedingstep, then after a sufficient number of cells attach to the at least onefiber and cell proliferation on the at least one collagen fiber to adesired level, the applying step applies a cyclical strain and/orstress.

The static strain can be carried out after cells introduced by theseeding step attach to the at least one elongate collagen fiber in adesired amount for a first time period, then the cyclic strain and/orstress is applied for between 5 minutes/day to 24 hours/day for asubsequent time period.

The method can include, prior to the placing step, winding, twisting,braiding or weaving a plurality of the elongate collagen fibers into acollagen fiber construct at a defined tension t_(f). The applying stepcan hold opposing end portions of the collagen fiber construct in thepouch at the tension t_(f).

The applying step, at least initially, can include applying a first lowstrain and/or tensile load to the collagen fiber construct, thenapplying cyclic strain or tension that varies between 2% to about 15%above the first low strain or tensile load.

The cycling the tensile load and/or strain can be carried out after theseeding step.

The cyclic strain can be applied to the at least one elongate collagenfiber for between about 5 minutes to about 24 hours per day for adefined number of days and/or until the cells differentiate sufficientlyand/or produce a desired amount of the extracellular matrix of collagenon the at least one collagen fiber.

The cyclic strain can vary between about 2% to about 10% above theinitial strain and cycles at between about 0.5 Hz to about 3 Hz for adefined time period of at least 30 minutes every 24 hours.

The strain and/or stress can be uniaxial.

The at least one fiber can be a plurality of fibers arranged as animplantable collagen fiber construct. The construct can have a tensilestrength, stiffness, and dynamic flexibility that meets or exceeds thatof a natural tissue the implantable collagen fiber construct is designedto mimic.

The tissue can be a tendon or a ligament prosthesis.

Other embodiments are directed to medical kits. The kits include asingle-use flexible package holding a collagen construct comprising aplurality of elongate collagen fibers. The collagen fibers includeligament or tendon phenotype cells, and the tendon or ligament phenotypecells extend over at least 50% of a length of the collagen fibers.

The elongate collagen fibers of the construct can also include anextracellular matrix comprising collagen, with the collagen present inan amount greater than other extracellular matrix proteins.

The flexible package can include a polymeric pouch. The kit can includean autotensioner releasably attached to the package to apply amaintenance strain and/or stress to the tissue.

The collagen fibers can be cross-linked with NDGA and the tendon orligament phenotype cells cover at least a major portion of a surfacearea of each of the collagen fibers for at least 50% of the length ofthe collagen fibers.

The plurality of elongate collagen fibers can be wound, braided, twistedand/or woven together.

The collagen construct can be a tendon or ligament prosthesis and has atensile strength, stiffness, and dynamic flexibility that meets orexceeds that of a natural tissue the collagen construct is designed tomimic.

The cells can be differentiated cells derived from cells selected fromthe group consisting of embryonic, neonatal or adult cells, pluripotentstem cells, mesenchymal stem cells, tendon and/or ligament fibroblasts,or combinations thereof.

The plurality of collagen fibers can be between 2-1000 fibers.

Yet other embodiments are directed to medical materials that include atleast one elongate synthetic collagen fiber with a plurality of cellsattached to the elongate collagen fiber. The cells comprise definedphenotype cells or the cells comprise defined phenotype cells and anextracellular matrix comprising collagen, wherein collagen is present inan amount greater than other extracellular matrix proteins.

The at least one collagen fiber can include the defined phenotype cellsand the extracellular matrix comprising collagen that increases a volumeof the fiber, measured dry, by between about 20-200%.

The defined phenotype cells and extracellular matrix of collagen extendover at least 50% of a length and about at least a major portion of anouter surface of the at least one elongate collagen fiber.

The at least one collagen fiber includes the defined phenotype cells andat least 20% extracellular matrix of collagen by volume, dry.

The defined phenotype cells can be phenotype cells of natural (human orveterinary) tissue targeted for treatment using the at least onecollagen fiber.

Other embodiments are directed to systems for mechanically applyingstrain and/or stress. The systems include: (a) a plurality ofcooperating holding members adapted to attach to opposing end portionsof a plurality of pouches enclosing respective collagen constructs; (b)at least one fluid source in fluid communication with the pouches, theat least one fluid source comprising a first fluid source with cells;and (c) at least one tensioner with an automated stroke cycle incommunication with the holding members.

The system can also include a controller and user interface incommunication with the tensioner. The user interface and controller canbe configured to allow a user to select a cyclic stroke cycle of thetensioner to control stress/strain applied to the pouches.

The at least one fluid source can include a second fluid source in fluidcommunication with the pouches, the second fluid source comprising cellculture media devoid of cells.

The system can include an incubator with a housing that encloses thepouches, wherein the incubator can control one or more of a temperature,humidity, and/or gaseous atmosphere the pouches are exposed to in theincubator housing.

The system can include a flow control system in communication with theat least one fluid source and the controller. The controller can controloperation of the flow control system to selectively direct when to flowfluid from the first fluid source to one or more of the pouches.

It is noted that aspects of the invention described with respect to oneembodiment, may be incorporated in a different embodiment although notspecifically described relative thereto. That is, all embodiments and/orfeatures of any embodiment can be combined in any way and/orcombination. Applicant reserves the right to change any originally filedclaim and/or file any new claim accordingly, including the right to beable to amend any originally filed claim to depend from and/orincorporate any feature of any other claim or claims although notoriginally claimed in that manner. These and other objects and/oraspects of the present invention are explained in detail in thespecification set forth below. Further features, advantages and detailsof the present invention will be appreciated by those of ordinary skillin the art from a reading, of the figures and the detailed descriptionof the preferred embodiments that follow, such description being merelyillustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a cell attached to a collagenconstruct according to embodiments of the invention.

FIG. 2A is a digital photograph of a portion of a collagen constructcomprising a ribbon with an integrated suture according to embodimentsof the invention.

FIG. 2B is a digital photograph of a portion of a collagen constructcomprising a narrower ribbon with an integrated suture similar relativeto the ribbon shown in FIG. 2A according to embodiments of theinvention.

FIGS. 3A and 3B are digital photographs of collagen constructscomprising ribbons with pairs of integrated sutures on each long side ofthe ribbon according to embodiments of the invention.

FIG. 3C is a digital photograph of collagen constructs comprisingribbons with no sutures according to embodiments of the invention.

FIG. 3D is a digital photograph of collagen constructs comprising atleast one elongate collagen fiber according to embodiments of theinvention.

FIG. 4 is a flow chart of operations that can be used to carry outembodiments of the invention.

FIG. 5A is a schematic illustration of a side perspective view of acollagen construct comprising an elongate collagen fiber according toembodiments of the invention.

FIG. 5B is a schematic illustration of a side perspective view of acollagen construct comprising a plurality of elongate collagen fibersaccording to embodiments of the invention.

FIGS. 6A-6C are schematic illustrations of implantable collagen devicesin position in a subject according to embodiments of the invention.

FIG. 7 is a graph of time (hour) v. strain and/or stress showing noapplication of strain and/or stress during seeding. The strain and/orstress is a prophetic example and the units of the strain and/or stressare nonspecific and used only to show the change in the strain and/orstress over time (hour).

FIG. 8 is a graph of time (hour) v. strain and/or stress showing varyingamounts of strain and/or stress for varying amounts of time afterseeding. The strain and/or stress is a prophetic example and the unitsof the strain and/or stress are nonspecific and used only to show thechange in the strain and/or stress over time (hour).

FIG. 9 is a graph of time (hour) v. strain and/or stress showing noapplication of strain and/or stress until 12 hours after seeding. Thestrain and/or stress is a prophetic example and the units of the strainand/or stress are nonspecific and used only to show the change in thestrain and/or stress over time (hour).

FIG. 10 is a graph of time (hour) v. strain and/or stress showingapplication of a static strain and/or stress during seeding. The strainand/or stress is a prophetic example and the units of the strain and/orstress are nonspecific and used only to show the change in the strainand/or stress over time (hour).

FIG. 11A is a schematic illustration of a device that can providetension to a collagen construct while seeding and/or culturing accordingto embodiments of the invention.

FIGS. 11B and 11C are schematic illustrations of alternate embodimentsof a device that can provide tension to a collagen construct whileseeding and/or culturing according to embodiments of the invention.

FIGS. 12A-12C are a schematic illustration of a series of steps that canbe carried out to prepare an implantable collagen device according toembodiments of the invention.

FIGS. 13A-13C are a schematic illustration of a series of steps that canbe carried out to prepare an implantable collagen device according toembodiments of the invention.

FIG. 14A is a schematic illustration of a collagen fiber constructplaced in a flexible package or bag/pouch according to embodiments ofthe present invention.

FIG. 14B is a schematic illustration of different loading configurationsthat can be used to apply a static and cyclic tensile loading, stress orstrain with an associated exemplary timing diagram according toembodiments of the present invention.

FIG. 15A is a top view of a cyclic autotensioner according toembodiments of the invention.

FIG. 15B is a side perspective view of an alternate embodiment of acyclic autotensioner according to embodiments of the invention.

FIG. 15C is a schematic illustration of medical kit comprising anelectric motor according to embodiments of the invention.

FIG. 16 is a schematic illustration of a system that can provideflow-through fluid while seeding and/or culturing multiple discretecollagen fibers or collagen fiber constructs according to embodiments ofthe present invention.

DETAILED DESCRIPTION

The present invention now is described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Broken lines illustrate optional features oroperations unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP §2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. As used herein, phrases suchas “between X and Y” and “between about X and Y” should be interpretedto include X and Y. As used herein, phrases such as “between about X andY” mean “between about X and about Y.” As used herein, phrases such as“from about X to Y” mean “from about X to about Y.”

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention. The sequence of operations (orsteps) is not limited to the order presented in the claims or figuresunless specifically indicated otherwise.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration (e.g., the value of the strain and/orstress) and the like, is meant to encompass variations of ±5% or less,such as 5%, 4%, 3%, 2%, 1%, 0.5%, or even 0.1% of the specified amount.

The term “sterile” means that the device or component meets or exceedssterility guidelines for medical use.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety.

Implantable Collagen Devices

The present invention is directed to implantable collagen devicescomprising, consisting essentially of, or consisting of a collagen fiberconstruct and at least one cell, wherein the at least one cell isattached to the collagen fiber construct. Typically, the at least onecell is differentiated into a plurality of tendon and/or ligamentphenotype cells.

The term “cell” as used herein refers to a cell from any source (e.g.,human or animal; living or cadaveric) and can be an allogeneic cell(e.g., a cell from a source that is the same species as thesubject/recipient of an implantable collagen device of the presentinvention), an autologous cell (e.g., a cell from the subject/recipientof an implantable collagen device of the present invention), and/or axenogeneic cell (e.g., a cell from a source that is a different speciesthan the subject/recipient of an implantable collagen device of thepresent invention). Exemplary cells include, but are not limited to,embryonic stem cells; determined stem cells; perinatal stem cells;committed progenitors; pluripotent stem cells such as inducedpluripotent stem cells; multipotent stem cells such as mesenchymal stemcells and hematopoietic cells; stems cells from adult tissues or cordblood; adult cells of any suitable type such as smooth muscle cells,cardiac muscle cells, epithelial cells, endothelial cells, urothelialcells, fibroblasts, myoblasts, chondrocytes, chondroblasts, osteoblasts,osteoclasts, keratinocytes, hepatocytes, bile duct cells, pancreaticislet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary,ovarian, testicular, salivary gland cells, and adipocytes; tumor cells;and any combination thereof. In some embodiments of the presentinvention, the stem cells can be obtained without destruction of a humanembryo.

One or more cells can be used in the methods and devices of the presentinvention and when more than one cell is used, the cells can be the sameor different. In certain embodiments of the present invention, the atleast one cell comprises a mesenchymal stem cell from any source and/ora fibroblast from any source. In some embodiments of the presentinvention, the at least one cell is a fibroblast from human foreskinand/or a fibroblast from an autograft tissue such as dermis. The term“attach”, “attachment”, and grammatical variants thereof, as usedherein, refer to cellular adhesion and/or binding to a collagen fiberand/or collagen construct. In some embodiments of the present invention,a cell 70 can attach to a collagen construct 60 and/or fiber 60 f via acell adhesion molecule 70 a, such as, but not limited to, selectins,integrins, and/or cadherins, thereby forming an implantable collagendevice 60 (FIG. 1).

“Collagen construct,” as used herein, refers to a device and/or materialthat comprises collagen. The collagen construct can be in finished orfinal form for use or in an unfinished or pre-final form. The collagenconstruct can comprise natural collagen, natural collagenous tissue,synthetic collagen, and/or any combination thereof. “Synthetic collagen”as used herein, refers to collagen material that has been formed and atleast one of chemically or physically created or altered from itsnaturally-occurring state into an elongate collagen fiber. In someembodiments of the present invention, a collagen construct can be for atendon or ligament repair. In other embodiments, the collagen constructcan be for skin or other tissue repairs or treatments.

Exemplary collagen constructs, include, but are not limited to, collagenfiber patches, such as wound bed patches, muscle or organ patches,cardiac patches, hernia patches, skin patches, burn treatment patches,and skin/tissue repair patches; cuffs; blood vessel (artery, vein, andthe like) repair material; valve replacements or valve repair material;auto-graft material; allo-graft material; xenograft material; nerveguides; tubes; tendon sleeves, such as sleeves that can reside aboutrepairing tendon to prevent or inhibit adhesions; indwelling tubes fordelivery of therapeutic agents; ducts, such as lymphatic, hepatic,pancreatic and cystic ducts; tubes, such as ureter and urethra tubes;collagen fiber; collagen gel; sutures; cords; twisted cords; ligamentand/or tendon prosthesis; cables; braids; ribbons; staples; rivets;sponges; and the like. Further examples and description of devices aredescribed in U.S. Pat. No. 7,901,455; U.S. Patent ApplicationPublication Nos. 2008/0161917, 2008/0188933, 2008/0200992, 2009/0216233,2009/0287308, 2010/0094318, and 2010/0094404; U.S. patent applicationSer. Nos. 13/153,665 and 13/105,353; and U.S. Provisional PatentApplication No. 61/450,179, which are incorporated herein by referencein their entirety.

A collagen fiber or collagen fiber construct can be polymerized with asuitable cross-linking agent. A collagen construct can include elongatefibers that can be polymerized with a suitable cross-linking agent.Exemplary cross-linking agents include, but are not limited to,nor-dihydroguaiaretic acid (NDGA), 3,4-dihydroxyphenylalanine, dopamine,3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, carbodiimide,glutaraldehyde, formaldehyde, tannic acid, isocyanates, epoxy resins,and cross-linking agents comprising a quinone group and/or a catecholgroup, such as those described in U.S. Provisional Patent ApplicationNo. 61/530,115, on page 13, second paragraph to page 14, firstparagraph, this content is incorporated herein.

In certain embodiments of the present invention, the collagen fiberconstruct, such as, for example, braids, weaves, mandrel-wound fibers,cables, ribbons, staples, patches and rivets, is not polymerized with across-linking agent. The collagen fiber construct can comprise a tubethat comprises a partially dried collagen gel(s). In other embodiments,the collagen construct comprises a sponge.

In some embodiments of the present invention, the collagen constructcomprises at least one synthetic collagen fiber. A synthetic collagenfiber can be an elongate continuous length of fiber formed of denatured(gelatin) and/or non-denatured collagen (e.g., whole or fragmentednative collagen fibers from tendon, skin, or other sources). An elongatecollagen fiber can have a length of at least about 0.25 inches,typically greater than about 0.5 inches, such as between about 0.5-30inches, or between about 1-20 inches or between about 1 m to about 100m.

According to some embodiments of the present invention, a collagenconstruct comprises at least one elongate collagen fiber which canoptionally be polymerized with a suitable cross-linking agent. In someembodiments, the collagen construct comprises a plurality of elongatecollagen fibers, which can optionally be polymerized with a suitablecross-linking agent. In certain embodiments, the collagen constructcomprises at least one polymerized elongate collagen fiber.

Examples of fiber configurations include a single fiber, a plurality offibers, a fiber bundle, or a plurality of fiber bundles. The fibers orfiber bundles can be twisted, woven, braided or wound to define arespective twisted, woven, a braided or wound collagen fiber construct.The wound fiber refers to fiber or fibers that can be formed into aconstruct shape that can define be modified for implantation in adesired shape using a mandrel-wound length of collagen fiber(s). See,e.g., U.S. patent application Ser. Nos. 13/153,665; 12/576,435;12/576,423 and PCT/US12/27366 (U.S. Provisional Application Ser. No.61/450,179), the contents of which are hereby incorporated by referenceas if recited in full herein. In some embodiments of the presentinvention, the collagen construct is a woven “ribbon” construct of warpand weft fibers optionally comprising integrated sutures. Examples ofwhich are shown in FIGS. 2A, 2B, 3A, and 3B.

FIGS. 2A and 2B illustrate examples of ribbons 10 as the construct 60with collagen fibers 60 f and at least one integrated suture 20 thatextends beyond at least one end of the ribbon body 10 b, and typicallybeyond both ends of the ribbon body 10 b a distance of at least about 1inch. The at least one suture 20 can be woven into the weave of theribbon body 10 b without requiring any supplemental fixation membersproviding for a seamless smooth configuration. FIGS. 2A and 2Billustrate a single (multi-fiber) suture 20 that is woven into anaxially extending (lengthwise direction) center portion of the ribbonbody 10 b and extends substantially straight throughout the length ofthe ribbon body. Two or more (typically substantially) parallel suturesoriented to extend in the lengthwise direction may also be used insteadof the single suture shown. FIGS. 3A and 3B illustrate examples ofribbons 10 with collagen fibers 60 f and transversely spaced apart pairsof sutures 20 ₁, 20 ₂ and 20 ₃, 20 ₄ woven directly into the ribbon body10 b, one pair proximate each opposing outer long side of the ribbonbody 10 b. Further exemplary collagen constructs include those describedin U.S. Pat. No. 7,901,455 and U.S. Provisional Patent Application No.61/450,179, which are incorporated herein by reference in theirentirety.

The term “woven” means that the woven construct includes one or morewarp and one or more weft yarns, typically between about 10-100 warp andbetween about 1-10 weft yarns. The woven construct can be a ribbon 10 bthat can have any suitable number of yarns, any suitable number offibers 60 f in each yarn, and/or any desired number of picks/inch toform the braid pattern. In some embodiments, the ribbons 10 can have asubstantially repeating weft pattern with a weft yarn(s) having across-over (a frequency) of about every 0.1 mm to about every 25 mm,typically between about every 0.5-10 mm, and more typically in asubstantially repeating pattern with a cross over between about every1-5 mm.

Each yarn in a ribbon 10 b can be a single fiber 60 f (also known asfilament) yarn or multi-fiber (multi-filament) yarn. Some yarns 10 y mayhave more fibers than others in a respective ribbon 10 b or all theyarns may have the same number of fibers 60 f. The fiber 60 f or fibersin each yarn(s) may be twisted or untwisted, or combinations of twistedand untwisted may be used for each or respective different yarns withina single ribbon 10 b.

In some embodiments, the yarns can each have between about 1-100elongate continuous length collagen fibers (treated to improve strength,such as treated with NDGA), typically between about 2-20 fibers in eachyarn. That is, the collagen fibers 60 f can have a length sufficient toextend over substantially the entire length of the ribbon body. Theribbons 10 b may have between about 1-1000 yarns, typically betweenabout 3-100 yarns some of which are weft and some of which are warpyarns. For example, the ribbon body 10 b can include between about10-100 warp yarns of between about four to eight long NDGA treatedcollagen fibers 60 f. The yarn fibers may be twisted or braided togetherfor a respective yarn.

In some embodiments of the present invention, the collagen construct 10comprises elongate collagen fibers that are wound, woven or braided toform a construct that does not comprise an integrated suture.

FIG. 3C illustrates examples of ribbons as the collagen construct 10.The ribbons 10 b may, but are not required to, include an integratedsuture. In some embodiments, a woven or braided “ribbon” construct thatdoes not comprise a suture is polymerized with a suitable cross-linkingagent, such as, but not limited to, NDGA.

In some embodiments of the present invention, the collagen constructcomprises at least one continuous length elongate collagen fiber. FIG.3D illustrates a collagen construct that comprises an elongate collagenfiber 60 f that has been spooled.

FIG. 3D further illustrates that, in some embodiments of the presentinvention, a collagen construct can comprise a plurality of elongatecollagen fibers that can comprise a braided collagen fiber construct 60b, a woven collagen fiber construct 60 w, a knitted collagen fiberconstruct 60 k, a twisted collagen fiber construct 60 t, a woundcollagen fiber construct 60 x and a collagen fiber patch 60 p.

The term “patch” refers to a piece or segment of biomaterial that can beplaced on and/or affixed to target anatomical structure, typically softtissue, to treat, protect, repair and/or reinforce a target site. Thepatch can be any geometric shape but is typically substantially planarand may, in position, conform to the shape of underlying or overlyingtissue.

The term “implantable” and derivatives thereof, as used herein, meansthe collagen device can be inserted, embedded, grafted or otherwiseacutely or chronically attached or placed in or on a subject.

The present invention finds use in medical applications and medicalstudies. The term “medical” includes both human and veterinary uses. Insome embodiments of the present invention, the collagen device can beimplanted in a subject. In other embodiments of the present invention,the collagen device can be utilized in in vitro, ex vivo, and/or in vivoexperimental studies.

Suitable subjects for treatment using constructs of the presentinvention include, but are not limited to, avians and mammals. The term“avian” as used herein includes, but is not limited to, chickens, ducks,geese, quail, turkeys, pheasants, parrots, parakeets, macaws,cockatiels, canaries, and finches. The term “mammal” as used hereinincludes, but is not limited to, primates (e.g., simians and humans),non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas),bovines, ovines, caprines, ungulates, porcines, equines, felines,canines, lagomorphs, pinnipeds, rodents (e.g., rats, hamsters, andmice), and mammals in utero. In some embodiments of the presentinvention the subject is a mammal and in certain embodiments the subjectis a human. Human subjects include both males and females of all agesincluding fetal, neonatal, infant, juvenile, adolescent, adult, andgeriatric subjects as well as pregnant subjects.

Generally stated, one or more collagen fibers or a device with a bodyhaving one or more collagen fibers is populated (seeded) with cells,such as one or more of embryonic, neonatal or adult cells, pluripotentstem cells, mesenchymal stem cells, tendon and/or ligament fibroblasts,or combinations thereof. The (e.g., stem) cells proliferate to a desiredlevel on the one or more collagen fibers. Then, strain (and/or stress)is applied to the fibers/device or at least one fiber, the cellsattached to the fibers and transduce the mechanical strain (deformation)signal which induces them to differentiate into tendon (or ligament, forexample) fibroblasts.

Once the cells differentiate, they can proliferate and can also begin toproduce an extracellular matrix comprising predominantly collagen.Collagen production is governed by the cyclic strain (and/or stress).The collagen fiber or collagen fibers of the body of the device caninclude the starting collagen fiber or collagen fiber construct: plus 1)differentiated tendon and/or ligament phenotype cells; 2) differentiatedtendon and/or ligament phenotype cells plus a minor amount ofextracellular collagen matrix; 3) differentiated tendon and/or ligamentcells plus a relatively large amount of extracellular matrix ofcollagen. The extracellular matrix can be predominantly collagen and mayhave other molecules and/or proteins, in lesser amounts.

The phenotype of differentiated cells can be evaluated as is known tothose of skill in the art based on one or more of cellular, morphologicand/or molecular characteristics and/or expression markers. For example,stem cells are stellated in appearance but when differentiated intofibroblasts with tenocyte morphology, they elongate and align with adirection of collagen fibers and/or fibrils.

Examples of markers for a tendon phenotypic expression include Type Icollagen, or

Types I and III collagen, expression of scleraxis, and/or atranscription factor associated with the tendon fibroblast phenotype.

The end device or fiber(s) with the collagen fibers, differentiated(phenotype) cells and extracellular matrix can comprise about 95%collagen, dry weight.

Exemplary Methods of Preparing an Implantable Collagen Device

A further aspect of the present invention comprises methods of preparingan implantable collagen device (FIG. 4) comprising, consistingessentially of, or consisting of providing a collagen fiber construct 60(block 150), seeding the collagen construct 60 with at least one cell 70(block 160), and applying a strain and/or stress to the collagenconstruct 60 and/or at least one cell 70 (block 170), thereby producingan implantable collagen device.

In particular embodiments of the present invention, the strain and/orstress is applied until a desired cellular event(s) occurs. Differentstrains can be applied. For example a static load or strain can beapplied during a seeding and/or post-seeding period until a desiredamount of cell proliferation on the at least one collagen fiber hasoccurred, then a cyclic strain and/or stress can be applied to promoteextracellular matrix formation and to maintain cell differentiation(e.g., tendon or ligament phentotype fibroblasts and/or cells).

According to some embodiments of the present invention, the collagenconstruct 60 can comprise an elongate collagen fiber 60 f (FIG. 5A). Inother embodiments of the present invention, a plurality of elongatecollagen fibers can together form a collagen construct 60 (FIG. 5B). Insome embodiments of the present invention, a plurality of elongatecollagen fibers are wound, twisted, woven or braided together (FIGS. 2A,2B, 3A, and 3B). When a collagen construct comprises more than oneelongate collagen fiber, each or some of the elongate collagen fibersthat form the construct can be seeded with a cell(s) and/or subject to astrain and/or stress.

In some embodiments, the collagen fiber construct is configured to mimica natural tendon or ligament and can have substantially the same orgreater tensile strength and substantially the same elastic modulus.Thus, during cycling, the construct and/or fibers can impart properstrain onto the seeded “starter” cells to promote cellular changes intotendon (or ligament) phenotype cells such that the cells from the cellculture change into tenocyte (or ligocyte) morphology to have elongatedcells aligned with the axial direction of the fibers or fibrils.

An implantable collagen device can be used (e.g., implanted) on its ownor in combination with one or more implantable collagen device(s). Asshown in FIG. 6A, an implantable collagen device 60 having at least onecollagen fiber 60 f of any size or shape, can be implanted in a subjectas a tendon repair with a first end portion 65 a attached to a tissue 75via a suture 20 and a second end portion 65 b attached using a sutureanchor 20 a. In other embodiments of the present invention, one or moreimplantable collagen devices of any size or shape can be implanted nextto and/or on top of one another. As shown in FIG. 6B, a plurality ofimplantable collagen devices 60 ₁, 60 ₂, and 60 ₃, of any size or shape,are implanted in a subject as a tendon repair with a first end portion65 a attached to a tissue 75 via a suture 20 and a second end portion 65b attached using a suture anchor 20 a. The one or more implantablecollagen devices 60 can be sewn together, such as with an integratedsuture 20 (FIG. 6C). The embodiments shown in FIGS. 6A-6C are forexample only. Both ends of the construct 60 can be implanted, attachedor anchored in the same manner, e.g., using a suture, or in differentmanners as is known to those of skill in the art.

In preparing an implantable collagen device of the present invention, atleast one cell is seeded onto a collagen construct. The term “seed”,“seeding”, and grammatical variants thereof as used herein, refer to oneor more defined cells being contacted, placed on, and/or loaded onto acollagen construct. Before, during, and/or after the seeding step, thecollagen construct can be maintained under cell culture conditions.During and/or after the seeding step and/or culturing, the at least onecell can attach to the collagen construct. In particular embodiments ofthe present invention, a collagen construct is seeded with at least onecell, such as, for example, at least one mesenchymal stem cell from anysource and/or a fibroblast from any source.

At least one (e.g., a clonal cell), typically at least a few cells to100 s of cells, or 1000 s or more cells can be driven to reproduce toproliferate, populate and attach to fiber attachment sites so thatsubstantially all or at least a majority of available attachment sitesare filled with the “starter” cells. Non-attached cells can be washedaway or otherwise removed. The cells can attach to cover substantiallyan entire, or at least a majority of, the surface area of each elongatefiber.

Cell culture media suitable for the methods of the present invention areknown in the art and include, but not limited to, Dulbecco's ModifiedEagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium high glucose(DMEM-H), McCoy's 5A Modified Medium, and Medium 199. The cell culturemedium can be supplemented with additional components such as, but notlimited to, vitamins, minerals, salts, growth factors, carbohydrates,proteins, serums, amino acids, attachment factors, cytokines, hormones,antibiotics, therapeutic agents, buffers, etc. The cell culturecomponents and/or conditions can be selected and/or changed during themethods of the present invention to enhance and/or stimulate certaincellular characteristics and/or properties. Examples of seeding methodsand cell culturing methods are described in U.S. Pat. Nos. 5,266,480,5,770,417, 6,537,567, and 6,962,814 and Oberpenning, F., et al., De novoreconstitution of a functional mammalian urinary bladder by tissueengineering, Nature Biotechnology, 17, 149-155 (1999), which areincorporated herein by reference in their entirety.

The cell seeding and/or culturing can be carried out in a sterileenvironment using equipment and methods known in the art. In someembodiments of the present invention, the temperature of the cellseeding and/or culturing environment is between about 25° C. to about40° C. or any range therein, such as between about 30° C. to about 40°C. or between about 35° C. to about 40° C. In particular embodiments ofthe present invention, the temperature of the cell seeding and/orculturing environment is about 37° C. The cell seeding and/or culturingenvironment can be at atmospheric pressure, reduced pressure (e.g.,vacuumized pressure), high pressure, and/or any combination thereof. Inparticular embodiments of the present invention, the pressure of thecell seeding and/or culturing environment is atmospheric pressure. Insome embodiments of the present invention, the cell culturing and/orseeding steps are carried out in an atmosphere of between about 2% toabout 20% carbon dioxide (CO₂) or any range therein, such as betweenabout 5% to about 10% or between about 5% and about 15% CO₂. Inparticular embodiments of the present invention, the cell seeding and/orculturing are carried out in an atmosphere of between about 5% to about10% CO₂. Other gases, such as, but not limited to, nitrogen and/oroxygen, can be added to the cell seeding and/or culturing atmosphere. Insome embodiments of the present invention, one or more gases can be usedto obtain and/or maintain the desired atmosphere (e.g., to maintain thedesired oxygen and/or carbon dioxide levels).

In some embodiments of the present invention, the seeding step iscarried out two or more times, such as between 2-20 times, including 2,3, 4, 5, 6, 7, 8, 9 or more times, during the preparation of animplantable collagen device of the present invention. For example, acollagen construct can be seeded with at least one cell and the cell canbe cultured for a period of time. Then, the collagen construct can beseeded a second time with at least one cell that is the same as ordifferent than the first cell(s). In some embodiments of the presentinvention, a cell culturing step is not necessary after the seeding stepwhen a desired cell confluency is obtained on a collagen construct afterthe seeding step.

According to some embodiments of the present invention, during and/orafter seeding and/or cell culturing, a collagen construct and/or cell(s)can be subjected to a force loading. “Force loading” as used hereinrefers to a tensile, strain and/or stress load on a collagen constructand/or cell(s). The strain and/or stress can be a direct and/or indirectforce that is created by mechanical, fluid, and/or electrical means. Insome embodiments of the present invention, a collagen construct and/orcell is subjected to a stress or a strain. In embodiments of the presentinvention, a collagen construct and/or cell is subjected to a stress anda strain in any order and/or combination. Those skilled in the art willappreciate that a stress on a collagen construct and/or cell can cause astrain on the collagen construct and/or cell.

While embodiments of the invention contemplate the use of flexiblepackages, e.g., elastomeric and/or polymeric pouches to hold the atleast one collagen fiber or collagen fiber construct during the seedingand applying of strain/stress to induce the cellular events,conventional cell culture vessels may also be used for such purpose.

In some embodiments, as shown in FIG. 13E, the mounted construct(typically in the package/bag/pouch as shown in FIG. 14A/B) is pulledfrom each end to remove slack. An inter-clamp distance D₁ is defined ormeasured, and an initial relatively low offset strain Ti of a desiredamount, e.g., about 1% can be applied. Cyclic strain can then be appliedas shown in the timing diagram associated with FIG. 14B. The magnitudeof strain can vary between 2% and 10%. The construct can substantiallyalways be held under tension, going from the initial offset to a desiredstrain. So the cyclic tension goes from 1% to say 11% at a definedfrequency (such as those stated herein). The associated distancesbetween clamps D₂ and D₃ can be used to control the tension/strainduring cycling. One distance can apply the maxima cyclic strain Tc₁ ortension and the other D₃ can apply a lower cyclic strain or tension Tc₂that is still above the initial offset strain or tension Ti associatedwith distance D₁.

In some embodiments, the loading can be carried out to apply stress. Theconstruct is loaded to reach a defined percent of a known failure load,typically about 1% of its failure load (the actual load will differdepending on the size of the construct). Then additional load is appliedcyclically to increase and decrease loading at a defined frequency. Theapplied load can vary from 2% of failure load to about 50% of failureload. The frequency can be constant over a defined time or may vary,e.g., from 10 Hz to 5 Hz over different time periods during the cellularevents or for maintenance of the cell differentiation.

It is contemplated that the loading can be carried out to induce one ormore of three outcomes. The cyclic tension and/or strain can continueuntil at least one of the following outcomes occurs: (i) the constructis populated by cells that have been ‘differentiated’ into tendon orligament phenotype cells, typically with an extracellular matrix ofcollagen; (ii) a thickness of the construct or height of organizedcells/tissue is sufficient so as to increase a thickness of theconstruct by between 10-200%, e.g., the cells can increase the thicknessof the collagen fiber construct itself as cells populate, differentiateinto tendon or ligament phenotypes and/or organize and produce atendon-like tissue; or (iii) the construct has a large extracellularmatrix of collagen or predominantly collagen (more collagen than othermolecules and/or proteins), e.g., an amount greater than the ligament ortendon phenotype cells.

In some embodiments, tissue may be expected to grow over the entireconstruct between the points where it is clamped (if clamping is used tohold the device/provide the loading). It is contemplated that thephenotype cells and extracellular matrix of tissue can at least doublethe thickness of the construct. For example, a thickness or height oforganized cells/tissue on the collagen fibers is sufficient so as toincrease a thickness of the construct by between 10-200%, e.g., thephenotype cells and/or extracellular matrix can double or increase thethickness fiber construct itself as cells grow and organize and producea tendon-like tissue.

A tensile load, strain and/or stress can be uniaxial, biaxial,multiaxial, or any combination thereof. In particular embodiments, thestrain and/or stress is uniaxial. In some embodiments of the presentinvention, during the method of preparing an implantable collagen deviceof the present invention, the strain and/or stress can be in the samedirection throughout the fabrication. Alternatively, the strain and/orstress can change directions during the fabrication. For example, insome embodiments of the present invention, the strain and/or stress isuniaxial along the horizontal axis of a collagen construct throughoutthe method of preparing a collagen fiber and/or implantable collagenfiber device.

In some embodiments of the present invention, the strain and/or stressis uniaxial along a long or short axis, e.g., a long or short horizontalaxis of a collagen construct for a period of time, then changes touniaxial along the short or long vertical axis of the collagen constructfor a period of time.

The strain and/or stress can directly and/or indirectly contact a celland/or can be directly and/or indirectly transmitted to a cell. Thestrain and/or stress can cause strain and/or stress within a cell bodyand/or can result in and/or initiate cell signaling within a cell and/orbetween one or more cells and/or their environment. The strain and/orstress can cause changes to occur within a cell and/or the cellularmembrane (e.g., changes in the cytoskeleton and/or cytoplasm, clusteringof membrane receptors, etc.). The strain and/or stress can change,alter, and/or regulate cell to cell interactions, cellular interactionswith the extracellular matrix, and/or the extracellular matrix. In someembodiments of the present invention, the strain and/or stress can causea cell to change its phenotype (e.g., differentiate) and/or theextracellular matrix. In particular embodiments of the presentinvention, a strain and/or stress causes a cell to differentiate into aparticular type of cell (e.g., a fibroblast, etc.). In certainembodiments of the present invention, a strain and/or stress causes acell to form a tissue (e.g., a connective tissue, a muscle tissue,etc.).

Exemplary types of actions that can be used to provide the strain and/orstress include, but are not limited to, tension, pressure, compression,shearing, torque, elastic deformation, brittle deformation, ductiledeformation, or any combination thereof. In some embodiments of thepresent invention, during the method of preparing an implantablecollagen device of the present invention, the strain and/or stress canbe the same type throughout the method or can change during the method.In certain embodiments of the present invention, the strain and/orstress is applied in the form of a tensile strain and/or stress inand/or across a collagen construct and/or cell. In other embodiments ofthe present invention, the strain and/or stress is applied in the formof a pressure, such as, but not limited to, a hydrostatic pressure,and/or a compressive force on a collagen construct and/or cell. Incertain embodiments of the present invention, the strain and/or stressis applied in the form of a shear strain and/or stress, pressurizedfluid, and/or flow vectors. In particular embodiments, the shear strainand/or stress can be created by altering a flow of a fluid across thesurface of a collagen construct and/or cell.

The strain and/or stress can be static and/or cyclic. The term “static”,as used herein, refers to a strain and/or stress that is constant orsubstantially constant for a period of time. The term “substantiallyconstant”, as used herein, refers to a strain and/or stress that onaverage changes by less than 1% over a defined period of time, such asabout 1 minute, 5 minutes, 10 minutes, 1 hour, 24 hours, and the like.Those skilled in the art will appreciate that a static strain and/orstress can created by gradually or quickly increasing the strain and/orstress applied to a collagen construct and/or cell until the desiredlevel of strain and/or stress is achieved, then a constant orsubstantially constant strain and/or stress can be maintained for aperiod of time and after this period of time the static strain and/orstress can be gradually or quickly removed resulting in a decreasingstrain and/or stress.

In particular embodiments of the present invention, the strain and/orstress is cyclic for at least part of a fabrication period, particularlyduring and/or after seeding. The term “cyclic”, as used herein, refersto a strain and/or stress that varies (increases and decreases) inmagnitude by a defined amount over a defined period of time. A cyclicstrain and/or stress can cycle between about 0.1 Hz to about 10 Hz overa period of time (e.g., hours or days) or any range therebetween, suchas between about 0.5 Hz to about 8 Hz or between about 1 Hz to about 5Hz. In some embodiments of the present invention, the cyclic strainand/or stress cycles at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 over a period of time, e.g., persecond, minute, per hour or per day. In particular embodiments of thepresent invention, the cyclic strain and/or stress cycles at betweenabout 1 Hz or at about 4 Hz.

In some embodiments, an initial strain can be applied, e.g., a lowinitial offset strain of between about 0.5% to about 1%. Then, cyclicstrain can be applied for a defined time. The variation in magnitude ofa cyclic strain can be between a value above the initial offset strain,such as between 1%-2% to about 25% (e.g., the strain on the collagenconstruct and/or cell) or any range therein. In some embodiments, themagnitude of strain can vary between 2% to 12%, between about 5% toabout 20%, or between 2% to about 10% of the strain and/or stress. Insome embodiments of the present invention, the maximum variation inmagnitude of the strain is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%. In particularembodiments of the present invention, the change in magnitude of thecyclic strain can vary between 2% to 10%.

Stated differently, a cyclic tension can be applied so that theconstruct is always under tension in a defined time frame and cyclesbetween increased tension and a lower tension at a defined frequencyover a defined time period. In some embodiments, the tension canincrease to cycle between tensile loads that is between 1% above toabout 15% above an initial or “base” tensile load Ti (sufficient toremove slack), typically during a cell growth or maintenance period(s).In some particular embodiments, a cyclic tension load is applied so thattension increases by between about 2% to 11% from the base or definedinitial load at a defined frequency (which can be the same or changeover time) for a defined time period.

In some embodiments, stress can be applied. The construct can be loaded(held in tension) at a defined % of its failure load (the actual loadcan vary depending on construct size and configuration). Additional loadcan be applied cyclically. The additional applied load can vary frombetween 2% to about 50% of the failure load.

The static and/or cyclic strain and/or stress can be applied over adesired period of time. For example, a static and/or cyclic strainand/or stress can be applied over minute(s), hour(s), day(s), week(s),and/or month(s). In some embodiments of the present invention, a staticand/or cyclic strain and/or stress is applied throughout the method ofpreparing an implantable collagen device of the present invention. Inother embodiments of the present invention, a static and/or cyclicstrain and/or stress is applied intermittently and/or at regular timeperiods during the method of preparing an implantable collagen device ofthe present invention.

In some embodiments of the present invention, a collagen constructand/or cell are subjected to strain and/or stress prior to seeding thecell on the collagen construct. In particular embodiments of the presentinvention, a collagen construct can be formed under strain and/orstress. After formation of the collagen construct, the strain and/orstress on the collagen construct can be maintained, changed, or removeduntil seeding.

In some embodiments, after seeding, and typically after cell attachmentto the collagen fiber or fibers, a collagen fiber, construct and/or cellthereon can be subjected to a strain and/or stress. The strain and/orstress during seeding can be the same or a different type and/or amountof strain and/or stress as the strain and/or stress applied during theformation of the collagen construct. In particular embodiments of thepresent invention, seeding is carried out with the same type and/or thesame amount of strain and/or stress utilized during the formation of thecollagen construct. For example, in some embodiments of the presentinvention, the collagen construct is a collagen fiber braid or weave inwhich the collagen fibers are formed (woven or braided) under a defined(e.g., static) tensile load. Once the collagen fiber woven or braidedconstruct is formed, the same load can be used during formation (e.g.,braiding/weaving) can be applied to the collagen fiber construct. Insome embodiments, the same load is applied after seeding. In someembodiments, the same load is applied before seeding. In someembodiments, the same load is applied during seeding and for a timeafter seeding.

In some embodiments of the present invention, a collagen construct(e.g., a construct of one or more collagen fibers or just one or morecollagen fibers) is not under strain and/or stress during seeding (butis typically held in tension). After seeding, in some embodiments, nostrain and/or stress is applied to a collagen construct and cellproximate thereto for a defined time. This time can be between about 1hour to about 1 week. In particular embodiments of the presentinvention, after seeding, no strain and/or stress is applied to acollagen construct and cell until one or more cells attach to thecollagen construct and/or until a desired confluency is obtained on thecollagen construct. For example, as shown in FIG. 7 no strain and/orstress is applied to a collagen construct during seeding (t=0) and nostrain and/or stress is applied until a defined post-seed time, t=1,e.g., shown for example only, is about 1 hour after seeding.Subsequently at t=2, e.g., a static strain and/or stress can be appliedfor defined time, e.g., about 1 hour. The amount of the static strainand/or stress can be decreased to a lower static strain and/or stressfor about 2 hours. This pattern of strain and/or stress can be repeatedfor a desired amount of time. The lower strain and/or stress can beapplied for a greater than, less than, or equal amount of time comparedto the period of higher strain and/or stress. Thus, a collagen constructand/or cell proximate thereto can be exposed to varying amounts ofstrain and/or stress for varying amounts of time (FIG. 8).

In some embodiments, no strain and/or stress is applied to a collagenconstruct during seeding (t=0). Then, after an initial (static orcyclic) strain and/or stress is applied, either a lower static or cyclicstrain and/or stress is applied or no strain and/or stress is applied tothe collagen construct and/or cell. As can be seen FIG. 9, no strainand/or stress is applied to a collagen construct during seeding (t=0)and no strain and/or stress is applied until about 12 hours afterseeding (t=12). At about t=12 hours, a cyclic tensile load or strainand/or stress is applied for about 2 hours. Then at about t=14 hours nostrain and/or stress is applied. This pattern of strain and/or stresscan change and can be repeated for a desired amount of time.

In certain embodiments of the present invention, a collagen construct isunder a static or cyclic strain and/or stress during seeding (t=0).After seeding, a static or cyclic strain and/or stress or no strainand/or stress can be applied to the collagen construct and cell(s). Ascan be seen in FIG. 10, a static strain and/or stress is applied duringseeding (t=0) and this load can remain constant for a defined time,e.g., about 24 hours (t=24). As shown, at about t=24 hours, a cyclicstrain and/or stress is applied for about 1 hour. As shown, at aboutt=25 hours (t=25), a low static strain and/or stress can be applied.This or other pattern of strain and/or stress can be repeated for adesired amount of time.

In some embodiments of the present invention, a (static and/or cyclic)strain or stress is applied to a collagen construct and/or cellproximate thereto for between about 5 minutes to about 24 hours per dayfor between about two days to about 2 months. Typically, the applicationof a static and/or cyclic strain and/or stress will continue until ashort period prior to use, e.g., providing a shelf life of about 6 hoursto about 1 week, once stopped. In certain embodiments of the presentinvention, at least one cell (typically many cells) is seeded onto acollagen construct under a static strain and/or stress, then after aperiod of time sufficient for cell attachment and proliferation to adesired level, such as between about 5 minutes to about 48 hours, acyclic strain and/or stress is applied to the collagen fibers orcollagen construct and/or cell(s).

In particular embodiments of the present invention, a cyclic strainand/or stress is applied to a collagen construct and/or cell(s) forbetween about 5 minutes to about 4 hours per day for about two days toabout 2 months. In some embodiments of the present invention, a cyclicstrain and/or stress is applied to a collagen construct and/or cell(s)for about 10 to about 30 minutes per day, wherein the cyclic strainand/or stress varies in magnitude by between about 5% to about 15% andat between about 1 Hz to about 2 Hz, and for the remainder of the day,no strain and/or stress is applied to the collagen construct and/orcell(s) or a static load, e.g., static strain is applied to the collagenconstruct and/or cell(s).

In some embodiments of the present invention, a static and/or cyclicstrain and/or stress is applied to a collagen construct and/or cell(s)until a desired cellular event occurs (e.g., cell differentiation,change in the cellular morphology and/or the extracellular matrix,production of an extracellular matrix and/or a tissue that resemblesnormal tissue, such as a tendon, etc.). In certain embodiments of thepresent invention, a static and/or cyclic strain and/or stress isapplied to a collagen construct and/or cell(s) until a tissue is formedon the collagen construct. In particular embodiments of the presentinvention, the tissue formed on the collagen construct resembles atendon or a ligament tissue.

The term “tissue”, as used herein in reference to a tissue formed on acollagen construct, refers to a two or three dimensional mass of livingtissue that can be produced by in vitro and/or ex vivo cellular growthaccording to the methods of the present invention. A tissue formed on acollagen construct according to the methods of the present invention canalso be referred to as a tissue engineered construct. The tissue canform around and/or through a collagen construct. A tissue engineeredconstruct can comprise the same type of cells or different types ofcells. A tissue engineered construct can comprise one or more types oftissue (e.g., connective tissue, muscle tissue, epithelial tissue,etc.). Exemplary tissue engineered constructs include, but are notlimited to, a tendon, a ligament, a muscle, an organ, or any combinationthereof. In particular embodiments of the present invention, a tendon ora ligament is formed according to the methods of the present invention.In some embodiments of the present invention, a static and/or cyclicstrain and/or stress causes one or more cells to produce and/ordifferentiate to form a tendon or a ligament.

In certain embodiments of the present invention, the implantablecollagen device has a tensile strength, stiffness, and/or dynamicflexibility that meets or exceeds that of a natural tissue the device isdesigned to mimic, such as a tendon or ligament.

After formation of an implantable collagen device according to themethods of the present invention, the implantable collagen device can beimplanted in a subject within a defined shelf life of the device,typically within about two months. In certain embodiments of the presentinvention, the implantable collagen device can be implanted in a subjectabout 2 to 3 weeks after formation. In some embodiments of the presentinvention, a static and/or cyclic strain and/or stress can be applied toa implantable collagen device after formation until a short time beforeimplantation (e.g., less than about 1 week) to maintain thedifferentiated cell status of the attached phenotype cells on theimplantable collagen device and/or collagen fiber(s).

The methods of the present invention can be carried out in a packagedesigned to accommodate one or more steps of the methods of the presentinvention (e.g., seeding, culturing, strain and/or stress, and storage)and the package can comprise one or more collagen constructs, such as 2,3, 4, 5, or more. The package can be sealable via a seam, flap,adhesive, lid, cap, and/or the like. The package can be a flexiblepackage that can be configured to allow seeding and/or cell culturingand/or cooperate with a device to apply a strain and/or stress to acollagen construct and/or cell. Exemplary means for carrying out seedingand/or cell culturing include but are not limited to, a fluid port, agas port, a gas exchange port, a pressure valve, and the like. Thepackage can attach to a device to apply the strain and/or stress using,for example, but are not limited to, one or more of clamps, hooks,clips, staples, sutures, an electrical lead, a fluid port(s), a gasport(s), a pressure or vacuum chamber that holds the package (e.g.,flexible pouch) and/or the like.

In certain embodiments of the present invention, a means for applyingstrain and/or stress comprises a mechanical device that can applytension and/or compression. One or more end portions (typically twoopposing end portions) of a collagen construct held in a package isattached to a mechanical device such as a tension mechanism(“tensioner”), in order to apply tension, strain and/or stress to thecollagen construct.

In particular embodiments of the present invention, a package allows fora collagen construct to be placed in the package such that the collagenconstruct does not touch the sides of the package. In other embodimentsof the present invention, the collagen construct is freely mobile in thepackage and can optionally be fixed during application of a strainand/or stress. In some embodiments, opposing ends of the constructand/or fiber(s) are clamped against outerwalls of the package and thepackage and fibers/construct can move in concert during application ofstrain/stress. In some embodiments, the fiber(s) and/or construct areencased inside the package. In other embodiments, outer opposing ends ofthe at least one fiber or fiber construct can reside outside the packageand connect to an automated tensioner.

As shown in FIG. 11A, a collagen construct 60 can be placed in a vessel,e.g., a package 100 before, during, and/or after seeding. In certainembodiments of the present invention, a package 100 can cooperate with aholding member 50 such as a clamp 50 c that can attach to a collagenconstruct 60 contained within the package 100. The holding member 50 canattach to a tensioner device T that provides tension, strain and/orstress to a collagen construct 60 and/or cell 70. The package 100 can bea flexible pouch of one or multiple layers of material(s). The package100 can be translucent or transparent or comprise a transparent ortranslucent window to allow an optical or visual access to internalcontent to assess seeding and/or cell or tissue formation.

The package 100 can comprise at least one fluid port 80 to allow for theaddition and/or removal of cells and solutions, such as cell culturemedia. The fluid port 80 can be capped 80 c or otherwise sealed when notin use. At least one gas exchange port 85 can be included in thepackage. The gas exchange port 85 can be separate from the fluid portcap 80 c or be defined in part of the fluid port cap 80 c. A package cancomprise an indicator and/or label 87 showing the shelf life of theimplantable collagen device 60 in the package and/or the date and/ortime the implantable collagen device 60 in the package was prepared.FIG. 11B illustrates that the holding member 50 can comprise a hook andaperture 50 h, while FIG. 11C illustrates that the holding member 50 cancomprise an adhesive collar or band 50 a. The holding member 50 can haveother configurations including attachment can be via frictionalengagement, adhesive engagement, crimping, clamping, hooking, or othersuitable attachment means.

In some embodiments of the present invention, the package can be sterileand/or can provide a sterile environment for cell seeding, culturing,and/or construct storage or shipment. The interior surfaces of a packagecan be treated to prevent and/or inhibit cell adhesion to the interiorsurfaces of the package.

A package can comprise materials suitable for cell culture and/or forthe application of strain and/or stress. In some embodiments of thepresent invention, the package is a pouch of an elastomeric, polymericor other suitable flexible material such that it is substantiallyimpermeable and is capable of being stretched. In particular embodimentsof the present invention, a package is a single use, disposable package.In other embodiments of the present invention, a package is capable ofbeing reused and in some embodiments is capable of being sterilized bymethods known in the art.

According to some embodiments of the present invention, a method ofmaking an implantable collagen device comprises: placing a collagenconstruct in a package (e.g., a pouch, bag, container, vessel, etc.);optionally holding the collagen construct in a defined orientation inthe package; seeding the collagen construct with a plurality of cells;and applying a strain and/or stress to the at collagen construct toinduce the cells to organize into a tissue, thereby producing animplantable collagen fiber construct. In particular embodiments of thepresent invention, the sealable package comprises at least one fluidport and can optionally be flexible. In some embodiments of the presentinvention, the collagen construct comprises at least one elongatecollagen fiber. Seeding of the collagen construct in the package can becarried out via methods known in the art. In some embodiments of thepresent invention, seeding is carried out by dispersing or flowing afluid (e.g., cell culture medium) comprising a plurality of cells onto acollagen construct. Alternatively, a collagen construct can be submergedor immersed in a fluid comprising a plurality of cells. In someembodiments of the present invention, the method further comprisesremoving the fluid and adding a second fluid that optionally comprisescells.

As shown in FIGS. 12A-C, a method of making an implantable collagenfiber construct can comprise: placing at least one elongate collagenfiber 60 f in a sealable package 100 s having a fluid inlet port 80 iand a fluid exit port 80 e and then holding the at least one elongatecollagen fiber 60 f in a defined orientation in the package 100 s. Next,a fluid 90 comprising a plurality of cells 70 can be flowed into thefluid inlet port 80 i of the package 100 s to seed the at least oneelongate collagen fiber 60 f and then a strain and/or stress can beapplied to the at least one elongate collagen fiber 60 f. The sealablepackage 100 s can cooperate with a holding member 50 that can attach tothe collagen fiber 60 f contained within the package 100 s and theholding member 50 can attach to a tensioner device T that providesstrain and/or stress to a collagen fiber 60 f and/or the cells 70. Aftera certain period of time waste fluid 90 w can be collected from thefluid exit port 80 e and more fluid 90 can be added via the fluid inletport 80 i.

In certain embodiments of the present invention, prior to placing acollagen construct in a sealable package, a fluid optionally comprisingone or more cells can be present in the package. After the construct isplaced in the package, a strain and/or stress can be applied to theconstruct and/or cells. As shown in FIGS. 13A-C, a method of making animplantable collagen fiber construct can comprise: placing at least oneelongate collagen fiber 60 f in a sealable package 100 s having a fluidport 80, wherein the sealable package 100 s comprises a fluid 90comprising a plurality of cells 70 (FIG. 13A). Then, closing, typicallysealing the package 100 s and optionally holding the at least oneelongate collagen fiber 60 f in a defined orientation in the package 100s (FIG. 13B). The sealable package 100 s can cooperate with a holdingmember 50 that can attach to the collagen fiber 60 f contained withinthe package 100 s and the holding member 50 can attach to a tensionerdevice T that can provide strain and/or stress to a collagen fiber 60 fand/or the cells 70. Thus, a strain and/or stress can be applied to thecollagen fiber 60 f by attaching the holding member 50 to the collagenfiber 60 f and applying a strain and/or stress using the tensionerdevice T (FIG. 13C). The fluid 90 can be drained via the fluid port 80and a second fluid optionally comprising cells 70 can be added via thefluid port 80.

According to some embodiments of the present invention, upon placing acollagen construct 60 (e.g., at least one elongate collagen fiber 60 f)in a sealable package, the elongate collagen fiber can be modifiedphysically (e.g., braided, molded, twisted, etc.) and/or chemically(e.g., changing the chemical composition and/or oxidation state of thecollagen construct and/or adding agents or compounds to the collagenconstruct such as, but not limited to, compounds that aid with celladhesion to the collagen construct). In some embodiments of the presentinvention, the collagen construct 60 and/or fiber(s) 60 f is subjectedto tension after being placed in the sealable package and prior toseeding. The strain and/or stress can be applied after seeding (e.g.,after sufficient cell attachment).

In some embodiments of the present invention, the addition and/orremoval of a fluid from the package, such as during and/or afterseeding, can be done to minimize perturbation of the collagen constructand any attached cells. Removal of a fluid can include removing all or aportion of the fluid. In particular embodiments of the presentinvention, the fluid can be removed and/or changed after a desiredperiod of time (e.g., about every 12 hours, 24 hours, 2 days, etc.) andreplaced with a second fluid, which can optionally comprise one or morecells.

The amount of a fluid added to and/or maintained in the package isgenerally an amount sufficient to cover or submerge all surfaces of thecollagen construct (and/or collagen fiber(s)), while keeping the fluidlevel to a minimum to prevent slowing of the oxygen diffusion rate tothe cells. Alternatively, high levels of a fluid can be maintained in apackage for a desired period of time to provide a higher hydrostaticpressure to the cells and/or to slow the oxygen diffusion rate to thecells. As the cells multiply and/or form two dimensional and/or threedimensional structures, the amount of fluid required in the package tocover a collagen construct may increase. Further, the composition of afluid in the methods of the present invention can be the same or changeduring the methods of the present invention. In some embodiments of thepresent invention, a fluid is constantly being refreshed by continuallyadding and removing a portion of the fluid. During the addition and/orremoval of a fluid from the package, a strain and/or stress may or maynot be applied.

A further aspect of the present invention comprises a medical kit. Amedical kit of the present invention can comprise, consist essentiallyof, or consist of a package holding an implantable medical device of thepresent invention in finished or final form for use or in an unfinishedor pre-final form. In particular embodiments of the present invention, amedical kit comprises an implantable medical device comprising aplurality of cells attached to a collagen construct, and wherein thecells have organized into a tissue induced by strain and/or stressapplied to the collagen construct. The tissue can extend around andthrough the collagen construct. As shown in FIGS. 6A and 6B, animplantable medical device comprises a tissue 175 of tendon and/orligament phenotype cells and an extracellular matrix of collagen extendsabout one or more collagen fibers 60 f. In other embodiments of thepresent invention, a medical kit comprises a package holding animplantable medical device comprising a plurality of cells attached to acollagen construct, wherein one or more cells may or may not haveattached to the collagen construct and have not yet formed a tissue.

A medical kit of the present invention can comprise one or moreimplantable medical devices. A medical kit of the present invention canallow for strain and/or stress to be applied continuously orintermittently until or a time before implantation of the collagenconstruct. The strain and/or stress can be used to induce a certaincellular event and/or for maintenance of the implantable medical device.A maintenance strain and/or stress can be the same as or different thanthe strain and/or stress used to form and/or prepare the implantablemedical device.

In certain embodiments of the present invention, a medical kit comprisesa portable cyclic autotensioner T_(A) that can be used to apply strainand/or stress to one or more implantable medical device(s) viaattachment to the collagen construct(s) in their respective package(s)(FIG. 15). An autotensioner T_(A) can be used to prepare an implantablecollagen device and/or to maintain an implantable collagen device suchas during delivery or storage until implantation of the device. Themanual strain and/or stress can be applied to the one or more collagenconstructs via a rotational tensile strain and/or stress applied by arotational mechanism (FIG. 15A) or via a sliding mechanism used to applythe tensile strain and/or stress (FIG. 15B).

As shown in FIG. 15A, an autotensioner T_(A) holds a plurality ofpackages 100 with the respective collagen constructs 60. Threaded rods165 r can retract or extend rails 166. The autotensioner T_(A) can beused to maintain cell differentiation and/or cell viability duringstorage and/or shipment. The autotensioner T_(A) can hold one or morepackages, such as 2, 3, 4, 5, or more packages. As shown in FIG. 15B, anautotensioner T_(A) comprises a plurality of linear slides 168 inchannels 168 c on rails 166 ₁ and 166 ₂ and holds a package 100 with thecollagen constructs 60 or a plurality of separate packages with therespective collagen constructs 60. The linear slides 168 can allow asmaller cyclic strain and/or stress stroke cycle. As shown in FIG. 15C,an electric motor can be connected to the one or more packages 100 todirect the movement of an implantable medical device in an autotensionerT_(A) and can comprise a processor to provide the time and distance tothe electric motor.

In certain embodiments of the present invention, a medical kit comprisesone or more fluids that can be utilized to maintain a cell and/ortissue. A medical kit of the present invention can provide anenvironment suitable for storage of a cell and/or tissue untilimplantation, such as, but not limited to, providing a containedenvironment with a desired level of carbon dioxide.

Another aspect of the present invention comprises a mass productionsystem with a plurality of packages 100 horizontally and/or verticallystacked for applying strain and/or stress to a plurality of sealablepackages holding one or more collagen constructs 60. As shown in FIG.16, a system 300 for applying a tensile strain and/or stress to aplurality of sealable packages 100 s holding collagen constructs 60 cancomprise a plurality of cooperating holding members 50 attached toopposing end portions of a plurality of the packages 100 e enclosingrespective collagen constructs 60; at least one fluid source 250 incommunication with the packages 100 s, the fluid source 250 comprisingcells 70; and at least one tensioner T with an automated stroke cycle incommunication with the holding members.

The tensioner T can include or be in communication with a controller 260that allows a user to select the stroke cycle to apply to the collagenconstructs 60.

In certain embodiments of the present invention, the system comprises atleast two fluid sources, wherein the first fluid source 250 comprisescells 70 and is used to seed to the collagen constructs 60 and thesecond fluid source 255 comprises a fluid that does not contain cells.The second fluid source 260 can replace the first fluid source 250 aftera desired period of time (e.g., 12 hours, 24 hours, 2 days, etc.). Insome embodiments of the present invention, the first and second fluidsources alternate in supplying fluid to the collagen constructs. Ashared fluid path can be used to deliver both fluids to respectivestations/vessels held thereat as shown. Dedicated ports and paths mayalso be used (not shown).

In other embodiments of the present invention, the system comprises anincubator 280 with a housing 300 h that encloses the pouches. Anincubator can maintain and/or control the environment the packages areexposed to. In some embodiments of the present invention, an incubatormaintains/controls the temperature, humidity levels, and/or atmosphere(i.e., pressure and gas content, such as carbon dioxide percentage) thepackages are exposed to.

The system can include a flow control system 260 f in communication withthe at least one fluid source and the controller 260. The flow controlsystem can include, for example, valves, pumps and/or other flow controlmembers. The controller 260 can control operation of the flow controlsystem to selectively (automatically) direct when to flow fluid from thefirst fluid source to one or more of the pouches.

Embodiments of the invention will now be explained with respect to thefollowing non-limiting examples.

EXAMPLES Example 1

A plurality of elongate collagen fibers can be braided to form a braidedcollagen fiber construct while the fibers are held at a tension t_(f1).The braided collagen fiber construct can be placed in a sealable,flexible bag and secured in a defined orientation in the bag. The bagcan then be sealed (except for one or more ports where used) andconnected to an automated tensioner via a set of cooperating holdingmembers, which can hold the opposing ends of the bag. Once connected,the tensioner can be manually or automatically set to apply a statictensile load to the collagen construct that is substantially if nottotally the same as the tension t_(f1) used to braid the collagenfibers.

A cell culture medium containing human mesenchymal stems cells and/orfibroblasts can be added to the bag before, after or during theplacement of the collagen fiber construct in the bag. In someembodiments, the cell culture medium can be flowed into the bag using aport and defined flow path from a culture source. The cell culturemedium can be added until there is a sufficient quantity to cover allsurfaces of the braided collagen fiber construct with the medium. Thequantity can be such that a volume of the medium in the bag is held to aminimum to minimize slowing of the rate of oxygen diffusion to thecells. After the addition of the cells, the bag can be kept understandard cell culture conditions. The static tensile load t_(f1) can bemaintained via the tensioner for about 24 hours or until a majority ofthe cells have attached to the braided collagen fiber construct.

After approximately 24 hours or after a majority of the cells haveattached to the braided collagen fiber construct, a cyclic tensile loadof about 5% to about 10% strain of t_(C1), which is greater than orequal to t_(f1), at about 1 to about 4 Hz can be applied to the braidedcollagen fiber construct for about thirty minutes each day for about 4days to about 2 weeks. For the remainder of the day, no tension orstress/strain can be applied to the braided collagen fiber construct.

Example 2

A plurality of elongate collagen fibers can be braided to form a braidedcollagen fiber construct or woven to form a woven collagen fiberconstruct, while the fibers are held at a tension t_(f2). The woven orbraided collagen fiber construct can be placed in a sealable, flexiblebag, which contains cell culture medium and human mesenchymal stemscells and/or fibroblasts. The woven or braided collagen fiber constructcan be secured in a defined orientation in the bag and the bag can besealed. The bag can be kept under standard cell culture conditions. Notension or a light tension (which does not apply more than a di minimisstress or strain) can be applied to the braided collagen fiber constructfor between about 12 to about 24 hours.

After sufficient cell attachment, the tensioner can be set to apply acyclic tensile load to the braided collagen fiber construct of about 1%to about 5% strain of t_(C2), which is greater than or equal to t_(f2),at about 1 to about 2 Hz for about 2 hours each day for about 4 days toabout 2 weeks. For the remainder of the day during this timeframe, astatic tensile load of about 1% of t_(f2) can be applied to the braidedcollagen fiber construct.

Example 3

An elongate collagen fiber construct can be formed under a tensilestrain and/or stress t_(f3) and once formed can remain under tensilestrain and/or stress t_(f3). The collagen fiber construct can be placedin a clean, typically sterile, flexible bag with inlet and exit portsand secured in a defined orientation in the bag. The bag can be sealed(with one or more optional intake and/or fluid exit ports) and/orconnected to flow paths of fluid sources and connected to a tensionervia a set of cooperating holding members, which can hold the opposingends of the bag.

Cell culture medium containing human mesenchymal stems cells and/orfibroblasts can be added to the bag until all surfaces of the collagenfiber construct are covered with the medium. The bag can then bepackaged with the tensioner comprising or in communication with acontroller, which can be set to maintain the tensile strain and/orstress t_(f3) for about 4 hours, then apply a cyclic tensile strainand/or stress at about 5% strain of t_(C3), which is less than or equalto t_(f3), at 1 Hz for about 1 hour each day for about 1 month. For theremainder of the day during this period, the tensile strain and/orstress t_(f3) can be resumed.

The bag can also be packaged with a fluid source connected via the inletport and the controller can be set to remove the fluid every 24 hoursvia the exit port and add new fluid comprising cell culture medium viathe inlet port. The bag with the tensioner and fluid source can then beshipped to a facility for implantation in a subject. The inlet andoutlet ports can be combined as a single port or more than two ports.

Example 4

An elongate collagen fiber construct can be formed under a tensilestrain and/or stress t_(f4) and once formed can remain under tensilestrain and/or stress t_(f4). The collagen fiber construct can then beplaced in a sealable, flexible bag with inlet and exit ports and securedin a defined orientation in the bag. Next, the bag can be sealed andconnected to a tensioner via a set of cooperating holding members, whichcab hold the opposing ends of the bag.

Cell culture medium containing human mesenchymal stems cells and/orfibroblasts can be added to the bag until all surfaces of the collagenfiber construct are covered with the medium. The tensile load t_(f4) canbe maintained via the tensioner for about 24 hours. After approximately24 hours, a cyclic tensile load of between about 4% to about 8% strainof t_(C4), which is greater than or equal to t_(f4), at about 0.5 toabout 2 Hz can be applied to the collagen fiber construct for about 1hour each day for about 2 weeks or until a three dimensional cellstructure is formed. For the remainder of the day, a very low, buttypically no, strain/stress may be applied to the collagen fiberconstruct.

At about 2 weeks or once a three dimensional cell structure is formed,the bag can be packaged with the tensioner comprising a controller,which can be set to maintain the same strain and/or stress protocoldescribed above for about 1 week or until implantation in a subject. Thebag can also be packaged with a fluid source comprising cell culturemedium connected via the inlet port and the controller can be set toremove the fluid every 24 hours via the exit port and add new fluid viathe inlet port. The bag with an on-board tensioner (forming part of ashipment container) and fluid source can then be shipped to a facilityfor implantation in a subject.

Example 5

A construct formed with elongate collagen fibers can be placed in adefined orientation in a container, such as, but not limited to, a cellculture vessel or a sterile, flexible bag or pouch. Cell culture mediumcontaining stems cells and/or fibroblasts can be added to the containeruntil surfaces of the collagen fiber construct are covered with themedium.

Holding members of an automated tensioner, such as clamps or otherholding member configurations, can attach to each opposing end of thebag/construct. The holding members can be moved to pull the constructaxially to remove slack. An inter-clamp distance can be measured ordefined and an initial offset strain of about 1% is applied. Cyclicstrain can then be applied. The magnitude of strain can vary between 2%and 15%. The construct can substantially always be held under tensiongoing from the initial offset to the desired cyclic strain. The cyclictension can go from 1% to about 15% above the initial strain at adefined frequency.

The cyclic tension and/strain continue until at least one of thefollowing outcomes occurs: (i) the construct is populated byligament/tendon phenotype cells only, cells that have been‘differentiated’ into tendon/ligament phenotype cells; or (ii) theconstruct has the phenotype cells and an extracellular matrix that ispredominantly collagen.

The amount of phenotype cells and extracellular matrix can increasethickness of the construct by between 10-200%, e.g., the thickness candouble. [00.193] The phenotype cells, extracellular matrix or tissue ofsame can reside between the points of contact of the holders (e.g.,clamps) or over at least 50% of the length of the construct. Forexample, over at least 50% of a length of the construct the thickness orheight of organized cells/tissue can increase a thickness of theconstruct by between 50-200%, e.g., the cells double the thickness fiberconstruct itself as cells grow and organize and produce a tendon-liketissue.

Example 6

A construct formed with woven elongate collagen fibers can be placed ina defined orientation in a container, such as, but not limited to, acell culture vessel or a sterile, flexible bag or pouch. The constructcan simulate elastic modulus of a natural tendon for tendon replacementor repair. Cell culture medium containing stems cells and/or fibroblastscan be added to the container to contact the collagen fibers. Cyclicstrain can then be applied to the fibers. Because the construct mimics anatural tendon, it imparts proper strain to promote and cellular changesinto tendon phenotype cells such that the cells from the cell culturechange into tenocyte morphology to have elongated cells aligned with theaxial direction of the fibers or fibrils.

The magnitude of strain can vary between 2% and 15%. The construct cansubstantially always be held under tension going from the initial offsetto the desired cyclic strain. The cyclic tension can go from 1% to about15% above the initial strain at a defined frequency.

The cyclic tension and/strain continue until at least one of thefollowing outcomes occurs: (i) the construct is populated by targetphenotype cells only, cells that have been ‘differentiated’ into targettissue phenotype cells; or (ii) the construct has the phenotype cellsand an extracellular matrix that is predominantly collagen.

The amount of phenotype cells and extracellular matrix can increasethickness of the construct by between 10-200%, e.g., the thickness candouble.

For example, over at least 50% of a length of the construct thethickness or height of organized cells/tissue can increase a thicknessof the construct by between 50-200%, e.g., the cells double thethickness fiber construct itself.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

That which is claimed is:
 1. A method of processing an implantablecollagen fiber, comprising: a) placing at least one elongate collagenfiber in a desired orientation in a tissue culture vessel; b) seedingthe at least one elongate collagen fiber with a plurality of cells whilein the vessel; and c) automatically applying a strain and/or stress tothe at least one elongate collagen fiber in the vessel to cause at leastone of the following cellular actions: (i) induce the cells todifferentiate into tendon or ligament fibroblasts or tendon or ligamentphenotype cells; (ii) form an extracellular matrix of some or at least amajor portion of collagen; or (iii) both induce the cells todifferentiate into tendon or ligament fibroblasts or tendon or ligamentphenotype cells and form an extracellular matrix, wherein theextracellular matrix comprises collagen in amount greater than otherproteins.
 2. The method of claim 1, further comprising (d) producing animplantable collagen fiber construct with (i) the tendon or ligamentphenotype differentiated cells or (ii) the tendon or ligament phenotypedifferentiated cells and the collagen based extracellular matrix based,at least in part, on the applying step.
 3. The method of claim 1,wherein the tissue culture vessel comprises a flexible pouch that holdsthe at least one collagen fiber in the desired orientation.
 4. Themethod of claim 3, wherein the seeding step is carried out byintroducing a fluid comprising the plurality of cells into the pouch viaa fluid port of the flexible pouch.
 5. The method of claim 1, whereinthe cells of the seeding step are selected from the group consisting ofembryonic, neonatal or adult cells, pluripotent stem cells from anytissue source, mesenchymal stem cells, tendon and/or ligamentfibroblasts, or combinations thereof.
 6. The method of claim 1, whereinthe seeding step is carried by immersing or submerging the at least oneelongate collagen fiber into a fluid contained in the flexible pouch,wherein the fluid comprises the plurality of cells.
 7. The method ofclaim 1, wherein the applying step is carried out by attaching atranslating mechanical tensioner to opposing end portions of theflexible pouch.
 8. The method of claim 1, wherein the applying step iscarried out at least periodically after seeding.
 9. The method of claim1, wherein the applying step is carried out after a time sufficient toallow seed attachment to the at least one fiber, then at leastperiodically until implantation.
 10. The method of claim 1, wherein theapplying step is carried out to first apply a static strain or stress,then apply a cyclic strain or stress.
 11. The method of claim 1, whereinthe applying step is carried out to apply a static initial offset strainto the at least one elongate collagen fiber after the seeding step, thenafter a sufficient number of cells attach to the at least one fiber andcell proliferation on the at least one collagen fiber to a desiredlevel, the applying step applies a cyclical strain and/or stress. 12.The method of claim 11, wherein the static strain is carried out aftercells introduced by the seeding step attach to the at least one elongatecollagen fiber in a desired amount for a first time period, then thecyclic strain and/or stress is applied for between 5 minutes/day to 24hours/day for a subsequent time period.
 13. The method of claim 1,further comprising, prior to the placing step, winding, twisting,braiding or weaving a plurality of the elongate collagen fibers into acollagen fiber construct at a defined tension t_(f).
 14. The method ofclaim 13, wherein the applying step comprises holding opposing endportions of the collagen fiber construct in the pouch at the tensiont_(f).
 15. The method of claim 14, wherein the applying step, at leastinitially, comprises applying a first low strain and/or tensile load tothe collagen fiber construct, then applying cyclic strain or tensionthat varies between 2% to about 15% above the first low strain ortensile load.
 16. The method of claim 15, wherein, the cycling thetensile load and/or strain is carried out after the seeding step. 17.The method of claim 15, wherein the cyclic strain is applied to the atleast one elongate collagen fiber for between about 5 minutes to about24 hours per day for a defined number of days and/or until the cellsdifferentiate sufficiently and/or produce a desired amount of theextracellular matrix of collagen on the at least one collagen fiber. 18.The method of claim 15, wherein the cyclic strain varies between about2% to about 10% above the initial strain and cycles at between about 0.5Hz to about 3 Hz for a defined time period of at least 30 minutes every24 hours.
 19. The method of claim 1, wherein the applying step iscarried out to apply strain and/or stress that is uniaxial.
 20. Themethod of claim 1, wherein the at least one fiber is a plurality offibers arranged as an implantable collagen fiber construct, and whereinthe construct has a tensile strength, stiffness, and dynamic flexibilitythat meets or exceeds that of a natural tissue the implantable collagenfiber construct is designed to mimic.
 21. The method of any of claim 1,wherein the tissue is a tendon or a ligament prosthesis.